Project summary Integrated transcriptional regulatory circuits govern gene expression programs at the right place and right time. Mutations in transcription factors contribute to tumorigenesis. Despite the accumulation of transcription factor profiling, how different transcription factors co-regulate gene expression remains poorly studied. The research gap leads to the poor resolving power for polygenic conditions, especially cancer. In addition, the regulation network changes among different cells. In order to systematically and accurately dissect the transcriptional regulation network in normal tissue homeostasis and tumorigenesis, efforts must be made to firstly generate genetic models based on the genome-wide association study (GWAS) from human patients, secondly using advantageous single cell techniques to investigate the regulation of multiple transcription factors at chromatin, transcription and posttranscription levels in single cells. For F99 phase of the proposed research, Chi Zhang will continue working on the transcriptional regulation network of tissue stem cell quiescence, immune privilege and long-term maintenance of stem cell function. Through genetic knockout of two transcription factors individually and combinatorially in different cell lineages at different stages, Chi spatiotemporally investigated the functional role in vivo. The established single cell RNA-seq, ATAC-seq, Cut&Run ChIP-seq also allow Chi to dissect out the regulation network of Foxc1 and Nfatc1 at molecular level, including promoter enhancer looping, transcription factor binding, open chromatin dynamics, super enhancer identities, etc. Then using the established multiphoton system, Chi would be able to visualize the cellular activity of tissue stem cells after genetic manipulation. For K00 phase of the proposed research, Chi will apply the methodology in the context of glioblastoma. Considering it as one of the most progressive cancer with life expectancy of 14 to 16 month and existing GWAS data indicating the mutation of multiple transcription factors in human patients. Using the established glioblastoma mouse model in Dr. Parada lab, Chi will try to uncover the transcription regulation network of cancer stem cell, cancer heterogeneity and immune privilege. At the technical level, the use of genetically engineered mouse models, sophisticated genomic tools including scATAC-seq/scRNA-seq and live imaging should establish a blueprint for others to link transcriptional mechanisms to stem cell activities in live animals.